Summary: Micro RNAs (miRNAs) are a class of short non-coding RNAs that have been identified as potentially powerful tools for the study and treatment of many diseases, including ischemic stroke. Due to their conserved sequence, targeting specific miRNAs using synthetic anti-microRNA (anti-miR) reagents makes them attractive targets for drug development. We recently established that stroke and factors modifying stroke responses, such as social isolation, can modulate miRNAs, especially miR-141-3p. Hence, miR-141-3p represents a promising new molecular target for stroke therapy. Therefore, by developing therapeutics tailored to antagonize miR-141-3p, we hope to generate an effective therapy for stroke. Herein, we propose a multi- disciplinary project that applys modern technology to advance a promising stroke therapy that targets miR-141- 3p using novel next-generation anti-miR-based tools. In the past, we demonstrated that nanotechnology- delivered peptide nucleic acid (PNA)-based miRNA inhibitors can target miR-155 for lymphoma therapy. Unlike most nucleic acids, PNAs are synthetic DNA mimics in which the phosphodiester backbone is substituted with a neutral N-(2-aminoethyl) glycine backbone. PNAs can bind single-stranded targets with high specificity and affinity and are not susceptible to proteases, making PNAs ideal molecules for targeting miRNAs. To improve the effectiveness of anti-miR PNAs further, we will exploit a new class of PNA analogs designated gamma PNAs (?PNAs), which are conformationally pre-organized and so have advantageous binding and solubility properties that should increase their effectiveness as anti-miR agents. For delivery, we will employ poly (lactic- co-glycolic acid) (PLGA)-based nanoformulations. As proof of principle for our stable next generation ?PNA- based anti-miR-141-3p as a stroke therapeutic, we propose to test delivery and efficacy in stroke-based diseased mouse models. Here, we will pursue two independent specific aims: 1) Synthesis and quality control analysis of an array of ?PNA-based anti-miR-141-3p in cell culture-based assays; and 2) Test in vivo efficacy of ?PNA-based anti-miR-141-3p variants in a diseased mouse model. We have assembled an interdisciplinary team to achieve our goals, with expertise in nucleic acid chemistry, drug formulation, and disease biology.